Prolonged treatment with tyrosine kinase inhibitors (TKI) including imatinib (IMA) or nilotinib (NIL), induces severe disturbances of bone metabolism in patients with chronic myeloid leukaemia. As Vitamin D₃ (VD₃) is involved in the complex cycle of bone remodelling, the present study investigated in vitro, the influence of IMA and NIL on VD₃ metabolism i) in HaCaT cells and ii) in cultured outer root sheath keratinocytes (ORS-KC) from hair follicles of IMA treated children. Cells were incubated in the presence of IMA or NIL. Concomitantly, specific inhibitors were applied to analyze the inhibition of the VD₃ processing cytochrome P450 isoenzyme family by TKIs. In vitro, IMA and NIL significantly impaired the production of calcitriol in HaCaT and cultured ORS-KC cells from hair follicles of IMA treated children. For NIL, this inhibitory effect demonstrated a 4-fold increase. In HaCaT and ORS-KC, application of specific CYP450 inhibitors revealed that CYP27B1 was impaired by IMA and NIL leading to an intracellular accumulation of calcidiol. However, during TKI treatment, KC of IMA treated children revealed no differences in calcidiol and calcitriol levels. In conclusion, IMA and NIL interfere with the Vitamin D3 cascade due to their metabolism by CYP27B1.